MDP-1 (Magnesium-Dependent Phosphatase-1) has been discovered and partially characterized in this lab (Selengut and Levine, Biochemistry 39, 8315-8324, 2000). The mechanism by which this enzyme hydrolyzes phosphate had not been determined nor had the physiological substrate been identified; however, several reasonable mechanistic possibilities have been ruled out as have a wide variety of potential substrates.One mechanism that had not been ruled out was catalysis utilizing an aspartate nucleophile. A large family of aspartate-nucleophile enzymes exist and, like MDP-1, require magnesium for activity. This family, known as the haloacid dehalogenase (HAD) family, contains a large and diverse group of phosphatases and phosphomutases that, in the cases where the physiological substrates are known, hydrolyze small phosphorylated compounds. However, none of these phosphatases show significant homology to MDP-1 as measured by common sequence comparison algorithms such as BLAST or FASTA. The active site regions of several of these enzymes have been elucidated and three short sequence motifs are believed to be characteristic of this group. MDP-1 contains three corresponding sequences that, although not identical, show a high degree of similarity. Significantly, these regions are the most well-conserved stretches among the MDP-1's from seventeen species so far identified. To test the hypothesis that MDP-1 is a member of the HAD-family of phosphohydrolases and utilizes the same mechanism as these enzymes, a series of ten site-directed mutants was prepared. As expected, conversion of the aspartate corresponding to the HAD-family nucleophile to an asparagine reduced the enzyme activity by an order of magnitude. Other mutations resulted in less dramatic reductions in activity that were consistent with the hypothesis. Some mutations that were expected to reduce the activity had negligible effects, perhaps indicating important differences between MDP-1 and the other members of the family or possibly these are artifacts of the poor test substrate (para-nitrophenyl phosphate) used here in lieu of the (unknown) physiological substrate. Efforts to chemically label the nucleophilic aspartate by direct observation of the phosphoryl-enzyme intermediate or by reduction of that intermediate have been unsuccessful.We concluded from the alignment of the sequence motifs and the successful site-directed mutagenesis studies that MDP-1 is properly assigned as an aspartate-dependent phosphatase related to the HAD family of enzymes.